We propose to eluccidate the primary structure of band 3, the predominant polypeptide of human erythrocyte membrane. This component comprises roughly 25% of the membrane protein mass, contributing about 1 x 10 to the six copies per cell. It spans the membrane asymmetrically; its very hydrophobic behavior on isolation appears to reflect this adaptation for membrane insertion. It is a glycoprotein with its carbohydrate residues facing outside the cell. At its cytoplasmic pole, it has a binding site (or sites) for at least two glycolytic enzymes. Its function appears to be the facilitated diffusion of anions (chloride and bicarbonate in particular) across the membrane. It is our aim to correlate its structure with function in the context of its membrane integration. We have cleaved this polypeptide in situ to generate fragments which represent the outer-surface, cytoplasmic surface, and membrane-spanning regions. A 23K fragment, generated by S-cyanylation has been purified and 95% of its sequence of a total of 205 residues have been elucidated. We propose to complete the sequence of this 23K piece and to gegin sequence analysis of the adjacent cytoplasmic 16K fragment. This fragment is generated by cleavage with trypsin in situ of the band 3 polypeptide and can be purified on preparative PAGE in SDS. After completion of the 16K and 23K fragment sequence, the primary structure of the entire cytoplasmic domain of band 3 will be known.